The experiments were conducted under controlled conditions using plastic pots having lower diameter of 7.8 cm, upper diameter of 13.5 cm and 12 cm in height. The soil was collected from the top layer (0-20 cm) from the Botanical Garden of the university. Soil was air dried crushed and sieved through 2 mm filter autoclaved at 121 ℃ for 2 h. The soil was autoclaved to exclude soil pathogens and other microorganisms if any. The autoclaved soil was poured in pots and kept in the growth chamber. The pots were filled with 500 g uncontaminated soil and partially decayed compost (cow manure) (2:1) and was used as growing medium. The cow dung was added into the soil for better performance of earthworms. A subsample of the study soil before mixing with compost was analyzed for its physicochemical characteristics. The soil used for the experiment was sandy loam soil having pH 7.8 , EC (Electrical conductivity) (µS/cm) =184.25 , TDS (Total Dissolved Solids) (mg/kg) = 130 , N (Nitrogen) (mg/kg) = 103 , P (Phosphorus) (mg/kg) = 10.6 , K (Potassium) (mg/kg) = 0.343 , %OC = 0.894, Cd (mg/kg) = ND (not detected by AAS).The Cd treatment was given by using anhydrous CdCl₂ (Minimum assay: 95.0%) procured from Hi-Media laboratories. The CdCl₂ anhydrous was added to the soil to make different concentrations of Cd 0.50 mM, 0.75 mM, 1.00 mM, and 1.25 mM (i.e. 56 mg/Kg , 84 mg/Kg , 112 mg/Kg and 140 mg/Kg respectively). The various treatments given are as shown below:(1)C0 (Control): (Cadmium absence);(2)C1: (0.5 mM Cd);(3)C2: (0.75 mM Cd);(4)C3: (1.00 mM Cd);(5)C4: (1.25 mM Cd).Each Cd treatment was given in soils without as well as with earthworms (WTE = without, WE = with earthworms). Earthworms (3 earthworms per pot) were inoculated after seven days of Cd treatment and incubated for 7 d in soil with earthworms. The seeds after surface sterilization were sown in soil containing different concentration of Cd and earthworms in plastic pots. These pots were kept in seed germinator under controlled conditions i.e. 25 ℃ temperature and 16:8 h dark: light photoperiod (1700 lx) for 15 d. Seedlings were harvested after 15 d followed by washing with distilled water. The growth and biochemical analysis was done on these seedlings.

Increased Cd uptake in plants in presence of earthworms enhances the total antioxidative capacity, metal chelating compounds and content of other antioxidants in plants grown under metal polluted soils. Earthworms can improve plant growth by improving nutrient availability to plants through their vermicasting activity. Their role in modifying soil pH and increasing metal phytoavailability made their use ideal in phytoremediation of polluted soils. Increased uptake and accumulation of Cd in plants activates the antioxidative system of plants takes place by addition of earthworms to soil.

The gene expression for the key enzymes involved in organic acid metabolism was studied to understand the role of earthworms in organic acid metabolism in plants under Cd metal stress. It was observed that in comparison to control (C0) seedlings the expression of CS, SUCLG1, SDH and FH was enhanced 1.72, 1.58, 1.65 and 1.88 folds in seedlings given C4 treatment with 1.25 mM dose of Cd respectively . However, after supplementation of earthworms to Cd treated soils given C4 treatment resulted in further enhancement in expression of CS (2.53 fold), SUCLG1 (2.35 fold), SDH (2.13 fold) and FH (3.06 fold) .

Seeds of B. juncea (cv. RLC-1) were given pre-sowing treatment with 24-epibrassinolide (EBR) solutions (0 and 100 nM EBR/L) for 8 h. Petri-plates were lined with Whatman1 filter paper and were supplemented with different imidacloprid (IMI) concentrations (0, 150, 200, and 250 mg IMI/L). The EBR treated seeds were rinsed with distilled water and grown in Petri-plates supplemented with IMI solutions (three petri-plates for each treatment). The Petri-plates were kept in seed germinator (temperature = 25 ℃ , photoperiod = 16 h, light intensity = 175 µmol m ^-2 s^-1) and the seedlings were harvested 10 days after sowing for further analysis.

Seed soaking with 24-epibrassinolide recovers the impaired growth of B. juncea seedlings under imidacloprid stress by modulating the expression of genes encoding key enzymes including chlorophyllase, citrate synthase, succinyl Co-A ligase, succinate dehydrogenase, fumarate hydratase, malate synthase, phytoene synthase, chalcone synthase, and phenylalanine ammonialyase.

In the present study, as compared to control seedlings, the expression of gene CHLASE (encoding chlorophyllase) was observed to increase by 2.66-fold under IMI toxicity, but seed soaking with EBR significantly reduced the expression of CHLASE to 1.07-fold in the seedlings under IMI toxicity . Data analysis using two-way ANOVA and Tukey's HSD showed significant difference for CHLASE expression in B. juncea seedlings (FIMI p < 0.01, FEBR p < 0.01, FIMI * EBR p < 0.001). MLR analysis of the fold change in CHLASE expression also revealed the increased expression of gene with IMI toxicity and EBR application (positive betaIMI-value), whereas interaction between IMI and EBR was observed to be negative .Further, in comparison to control seedlings, the expression of PSY (encoding phytoene synthase) and CHS (encoding chalcone synthase) was significantly enhanced by 5.22 and 4.54-folds respectively in the seedlings raised from EBR treated as well as untreated seeds grown under IMI stress . Significant differences in expression PSY (FIMI p < 0.001, FEBR P<0.05) and CHS (FIMI * EBR p < 0.001) were observed after analyzing the data using two-way ANOVA and Tukey's HSD. MLR analysis of fold change in gene expression also revealed the role of EBR in modulation of gene expression of PSY and CHS. Concentrations of IMI as well as EBR were regressed positively on the fold change in gene expression of PSY and CHS, thus revealing enhanced expressions of these genes under both the treatments. Moreover, interaction between IMI and EBR was positive for PSY expression, whereas negative interaction was observed for the expression of CHS .In the present study, the expression of PAL was also observed to enhance significantly by 6.68-fold in the seedlings raised from EBR treated seeds and grown under IMI stress . After analyzing the data using two-way ANOVA and Tukey's HSD, significant difference in the expression of PAL was observed (FIMI p < 0.01, FEBR p < 0.01, FIMI * EBR P<0.05). MLR analysis of the fold change in gene expression also confirmed the role of EBR in increasing the PAL gene expression under IMI pesticide stress. Positive beta-regression coefficients were observed for IMI, EBR, and IMI * EBR .The expression of genes encoding the key enzymes involved in organic acid metabolism was also studied to understand the role of EBR in organic acid metabolism under IMI pesticide stress. It was observed that as compared to control seedlings, the expression of CS (encoding citrate synthase, 2.35-fold), SUCLG1 (encoding succinyl-Co-A ligase, 1.57-fold), SDH (encoding succinate dehydrogenase, 2.01-fold), FH (encoding fumarate hydratase, 1.57-fold), and MS (encoding malate synthase, 1.91-fold) were increased in B. juncea seedlings raised from untreated seeds and grown under IMI pesticide toxicity . However, seed soaking with 100 nM EBR and germinating them under IMI toxicity resulted in further enhancement in expression of CS (2.61-fold), SUCLGD1 (4.18-fold), SDH (2.55-fold), FH (3.73-fold), and MS (4.03-fold). Data analysis using two-way ANOVA and Tukey's HSD showed significant differences in the expression of CS (FEBR p < 0.01, FIMI * EBR p < 0.01), SUCLG1 (FEBR p < 0.001, FIMI * EBR P<0.05), SDH (FEBR p < 0.01), FH (FEBR p < 0.001), and MS (FEBR p < 0.001). MLR analysis showed that gene expression in seedlings under IMI stress as well as after the EBR seed treatment was increased as indicated by positive beta-regression coefficients. Whereas, negative interactions were noticed between IMI and EBR treatments for the expression of all genes studied related to organic acid metabolism.

After acclimation, the plants were grown independently in different light treatment chambers at 20 ± 0.2 &#8451 and 65 ± 2% humidity until the harvest date (35 days after light treatment). The white fluorescent light (70 ± 5 µmol/m²/s) was maintained for 12 h, and then each of the blue, green, red, and white lights was irradiated at 70 ± 5 µmol/m²/s for 4 h using LED arrays (DR LED Networks Co., Seoul, Republic of Korea). The spectral energy distribution of four different LED arrays was measured from 300 to 800 nm with a spectroradiometer (International Light, RPS-900, U.S.). Their maximum spectral wavelengths were 463 (blue), 518 (green), and 632 nm (red); the white LEDs had a broad spectrum. Irradiance was measured using a quantum sensor (LI-COR, LI-191, Lincoln, NE, U.S.). Water was supplied daily with top irrigation and a nutrient solution (Hoagland, pH = 5.9 ± 0.2, electrical conductivity = 1.2 dS/m) every 4 days until harvest.

A quantitation and principal component analysis biplot demonstrated that luteolin-7-O-glucoside (2), luteolin-7-O-glucuronide (3), and quercetagetin-trimethyl ether (8) were the highest polyphenols yielded under green light, and dicaffeoylquinic acid isomer (4), dicaffeoylquinic acid isomer (5), naringenin (7), and apigenin-7-O-glucuronide (6) were greatest under red light. Chlorogenic acid (1) and 1,2,6-trihydroxy-7,8-dimethoxy-3-methylanthraquinone (9) were produced in similar concentrations under both light types. The white and blue light appeared inefficient for polyphenol production.

The experiment was carried out in 2013. Leaves of five raspberry cultivars ('Polana', 'Polka', 'Tulameen', 'Laszka' and 'Glen Ample') were collected at the time of cultivation. Three organic and neighborhood conventional farms were used for experimental purposes. From one cultivar (one field plot), 3-4 plants were chosen, which were analyzed separately. One sample consisted of 10 leaves. The farm was treated as a replication. [organic farm no. 1 Localization: akroczym(52° 26″ N 20° 36″ E), Type of Soil: sandy middle soil IVa and IVb category (15% floatable particles) pH 5.5, Kind of Fertilizer: cow manure, Dose of Fertilizers and Time of Given: 35 t/ha one year before raspberry planting, Plant Protection System: Grevit 200 SL; organic farm no. 2 Localization: Zaluski (52° 37″ N 20° 22″ E), Type of Soil: sandy middle soil, sandy-clay IV category (20% floatable particles), pH 5.5, Kind of Fertilizer:cow manure, Dose of Fertilizers and Time of Given: 30 t/ha one year before raspberry planting, Plant Protection System: no protection; organic farm no. 3 Localization: Radzanow(51° 33″ N 20° 51″ E), Type of Soil: sandy middle soil IVa and III category (10% floatable particles), pH 6.0, Kind of Fertilizer:sheep manure, green manure, Dose of Fertilizers and Time of Given: 10 t/ha and 15 t/ha one year before raspberry planting, Plant Protection System: Bioczos 33 SL, Grevit 200 SL; conventional farm no. 1 Localization: Czerwinsk nad Wisla (52° 23″ N 20° 20″ E), Type of Soil: sandy-loamy middle soil IV and III category (20% floatable particles), pH 5.5, Kind of Fertilizer: Hydrocomplex 12-11-18; Superba 8-11-36, Dose of Fertilizers and Time of Given: (200 kg/ha, 150 kg/ha) in autumn a year before raspberry planting; 3 doses in time of cultivation, Plant Protection System: Signum 33 WG, Miros 20 SP; conventional farm no. 2 Localization: Czerwinsk nad Wisla (52° 23″ N 20° 20″ E), Type of Soil: sandy-loamy middle soil IV and III category (25% floatable particles), pH 5.5, Kind of Fertilizer: amonium nitrate, polyphosphate, magnesium sulphate, Dose of Fertilizers and Time of Given: in autumn a year before raspberry planting; 3 doses in time of cultivation, Plant Protection System: Calypso 480 SC, Miros 20 SP, Zato 50 WG; conventional farm no. 3 Localization: Czerwinsk nad Wisla(52° 25″ N 20° 23″ E), Type of Soil: sandy-clay middle soil II and III category (20% floatable particles) pH 6.0, Kind of Fertilizer:Rosafert 5-12-24-3, Dose of Fertilizers and Time of Given: 250 kg/ha in autumn a year before raspberry planting; 4 doses in time of cultivation, Plant Protection System: Calypso 480 SC, Miros 20 SP, Zato 50 WG].

Compared with conventional raspberry leaves, organic raspberry leaves were characterized by a significantly higher content of dry matter, total polyphenols, total phenolic acids, chlorogenic acid, caffeic acid, salicylic acid and quercetin-3-O-rutinoside; moreover, the organic leaves were characterized by higher antioxidant activity. Among examined cultivars, 'Polka' c. was characterized by the highest antioxidant status. However, raspberry leaves from conventional farms contained more total carotenoids, violaxanthin, alpha-carotene, beta-carotene, total chlorophyll and individual forms of chlorophylls: a and b.